Epidermal differentiation generates the impermeable stratum corneum (SC), a two-compartment system, composed of lipid-depleted corneocytes and lipid-enriched, extracelluar lamellar membranes. At the interface of these two compartments lies the lipid-bound envelope (LBE), a ceramide-enriched leaflet, that is covalently bound to the outer surface proteins of the cornified envelope. Since omega-hydroxy ceramides re both the predominant lipid constituents of the LBE, and the acylated-omega-OH ceramides are critical components of the extracellular lamellae, these unique lipid species, must be critical for the formation of a competent barrier. However, despite their apparent importance, neither the metabolic pathways leading to omega-OH ceramides, nor the regulation of their generation has been addressed. Moreover, their hypothesized role both in forming the LE and the lamellar membrane structures of the SC remain unresolved. Therefore, we hypothesize that the synthesis of omega-OH ceramides, through the activity of a cytochrome P450 omega-hydroxylase (CYP4A), with the subsequent generation of the lipid-bound envelope and acyl-ceramides, are tightly regulated by epidermal differentiation, permeability barrier requirements, and the program of epidermal barrier development in utero, with members of the nuclear hormone receptor family as the principle regulatory regulators. In this proposal, we will examine whether the generation of the unique epidermal omega-Oh ceramides is critical for formation of both the LBE and the lamellar membranes of the stratum corneum. Specifically, we will address: 1) the regulation of the key enzymatic step (i.e., omega- hydroxylation by epidermal CYP4A) leading to the generation of omega- OH ceramides during epidermal differentiation; 2) the regulation of , and requirement of, omega-OH ceramide synthesis for murine permeability barrier homeostasis; 3) the regulation of, and requirement for, CYP4A and omega-OH ceramide synthesis during fetal barrier ontogenesis; in addition, whether factors that accelerate epidermal barrier development modulate omega-OH ceramide generations; 4) the regulation of omega-hydroxylation and CYP4a expression by members of the nuclear hormone receptor family; and finally, 5) the interaction between the cornified envelope protein(s) and omega-OH ceramides in the formation of the LBE. Since the formation of the LBE, and subsequent generation of the extracellular lamellae, are late steps in epidermal differentiation, the proposed studies should result in significant new information regarding epidermal structure, function, and homeostasis.